In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle MHC II (NMHC II) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have been studying the transcriptional regulation of NMHC II-A and II-C genes as well as tissue-dependent regulation of alternative splicing of NMHC II-B and C genes. During the course of studying the regulatory mechanism for alternative splicing of NMHC II-B, we found that a new RNA binding protein family, the Rbfox family, plays a critical role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. In this report, we focus on the splicing regulators, Rbfox proteins. Rbfox proteins contain a single conserved RNA recognition motif (RRM) in the central region of the molecule and bind specifically to an RNA penta(hexa)nucleotide (U)GCAUG. There are three genes for Rbfox family proteins in mammals, Rbfox1, Rbfox2 and Rbfox3. Rbfox1 is expressed in brain and striated muscles whereas Rbfox2 is expressed in various tissues including brain and muscles. Notably, Rbfox3 expression is restricted to neural tissues. Biochemical analyses of brain cells sorted by Rbfox antibody staining and histological analyses demonstrated that the expression level of the neuron-specific splice variant of NMHC II-B mRNA correlated better with the level of Rbfox-3 expression rather than with that of Rbfox-1 or Rbfox2 expression. These observations suggest that Rbfox3 contributes neuron-specific splicing of NMHC II-B mRNA, although brain expresses all three Rbfox proteins. Next we extended our research to study the biological function of Rbfox3 using two model systems. First we made use of mouse embryonic carcinoma P19 cells which are capable of differentiating into neuronal cells following retinoic acid treatment. Neuronal differentiation of P19 cells can be monitored by outgrowth of a long axon-like extension which contains an axonal marker, phosphorylated neurofilaments. During neuronal differentiation, expression of Rbfox3 is induced whereas undifferentiated P19 cells do not express Rbfox3. Rbfox1 is barely detected under both undifferentiated and differentiated conditions and the Rbfox2 expression level is unchanged before and after differentiation. The shRNA-mediated knock-down of Rbfox3 results in a decrease in axon-like extensions and an almost complete elimination of phosphorylated neurofilaments. These results indicate that Rbfox3 is required for neuronal differentiation of P19 cells. Second we used the chicken embryonic spinal cord to study a role for Rbfox3 in neuronal development in vivo. SiRNA-mediated loss-of-function studies show that Rbfox3 is required to promote neuronal differentiation of postmitotic motor neurons as well as interneurons. Numb pre-mRNA encoding a signaling adaptor protein is found to be a target of Rbfox3 action, and Rbfox3 represses the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform by siRNA reproduces similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform is sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development.